Biologic and therapeutic relevance of DNMT3A mutations in acute myeloid leukemia Despite the many new advances in cancer research and treatment, therapeutic resistance remains the core challenge in clinical oncology. Genome sequencing efforts have made possible the identification of genes and specific mutations associated with therapeutic failure in the clinic. However, the molecular basis of therapeutic resistance remains largely enigmatic. Recurrent DNMT3A mutations are detected in 25-30% of acute myeloid leukemia (AML) patients and are associated with adverse outcome and resistance to frontline chemotherapy. DNMT3A mutations most often affect amino acid residue R882, and recent work has shown that these mutants display decreased enzymatic activity and aberrant binding properties. In addition, previous studies have shown that increased expression of DNMT3A functions as a pro-apoptotic switch in response to genotoxic stress. We hypothesized that DNMT3A mutations protect cells from apoptosis in response to DNA damage caused by oncogene activation, or by ionizing irradiation (IR) or chemotherapeutics, allowing for the survival of malignant hematopoietic cells. Our preliminary studies show that expression of the AML-associated DNMT3A mutants in primary hematopoietic cells inhibits myeloid differentiation, with accumulation of stem/progenitor cells, reduces apoptosis and increases self-renewal in vitro. In addition, cells harboring mutant DNMT3A exhibited hallmarks of genomic instability. Our mechanistic studies point at impaired DNA damage response including attenuation of p53 activation. This research proposal outlines a three-tiered approach to the comprehensive characterization of the role for DNMT3A mutations in normal and leukemic hematopoiesis and therapeutic resistance. First, we will use adoptive transfer approach in vivo studies and ex vivo studies to assess the contribution of leukemia-associated DNMT3A mutants to alterations in differentiation, apoptosis, and self-renewal in different stem/progenitor cell populations. Next, the molecular mechanisms underlying etiologic role of mutant DNMT3A in AML pathogenesis will be explored. We will address the changes in DNA methylation patterns and DNA binding in DNMT3A-mutant cells through genome-wide methylation profiling and ChIP-seq studies, and perform detailed analysis of DNA damage sensing, response and repair in cells with and without DNMT3A mutations. These studies will be initiated during the mentored phase, and will continue into the independent stage of the award, when the main focus will shift to pre-clinical development of mechanistically-informed anti-leukemic treatments. These functional, mechanistic and pre-clinical studies will be carried out in a novel genetic mouse model expressing leukemia-associated Dnmt3a mutant from its native locus. Overall we posit that genetic alterations in the DNMT3A gene play an important role in development of AML in many patients, and a more thorough understanding of this phenomenon is likely to have eventual diagnostic, prognostic and therapeutic implications.